Nitrogen fixation is a process in which N2 is reduced to NH4 ion by the enzyme complex, nitrogenase. One of the key questions in the study of N2 fixation at this time concerns its mode of regulation. Most previous studies on nitrogenase regulation have dealt with factors controlling its synthesis. We have found that in addition to controlling its synthesis, the photosynthetic bacterium Rhodospirillum rubrum can also regulate nitrogenase by controlling the activity of this enzyme. Preliminary data show that the cell controls this activity by converting an active unregulated form of the enzyme to a form that can be regulated. The conversion to the regulatory form occurs in response to changes in the nitrogen substrate of the cell; the addition of either glutamate or NH4 ion to the media causes the nitrogenase to be converted from the active to the regulatory form. Such a mechanism would give R. rubrum the ability to respond immediately to favorable changes in the nitrogen content of its environment and thus closely control the amount of energy consumed by this enzyme (10 to 15 moles of ATP are required to reduce 1 mole of N2). We have evidence suggesting that glutamine synthetase may play a role in this process. The goal of the proposed research is to understand the detailed mechanism of this new nitrogenase regulatory process. To study this regulatory mechanism utilizing modern biochemical techniques, we propose (1) to study in vitro the mechanisms by which nitrogenous compounds convert active nitrogenase to the regulatory form in R. rubrum; (2) determine if NH4 ion- and glutamate-mediated regulation of nitrogenase is controlled through the action of glutamine synthetase; (3) purify and characterize a protein activating factor required for activity of the regulatory form of nitrogenase; (4) compare chemical and physical properties of the active and regulatory forms of the nitrogenase; and (5) determine how widespread nitrogen substrate-mediated regulation of nitrogenase is among N2 fixing organisms.